Yoko Hayashi

Immunoaffinity purification of 11-dehydro-thromboxane B2 from human urine and plasma for quantitative analysis by radioimmunoassay

11-Dehydro-thromboxane B2 is now considered to be a reliable parameter of thromboxane A2 formation in vivo. An immunoaffinity purification method was developed for radioimmunoassay of this compound contained in human urine and plasma. Monoclonal anti-11-dehydro-thromboxane B2 antibody was prepared and coupled to BrCN-activated Sepharose 4B. Human urine or plasma was applied to a disposable column of the immobilized antibody. After the column was washed with water, 11-dehydro-thromboxane B2 was eluted with methanol/water (95/5) with a recovery of more than 90%. The purified extract was subjected to a radioimmunoassay utilizing 11-[3H]dehydro-thromboxane B2 methyl ester and the monoclonal anti-11-dehydro-thromboxane B2 antibody. The detection range of the assay was 10-600 fmol (IC50 = 90 fmol). The cross-reactivities of the antibody with thromboxane B2, 2,3-dinor-thromboxane B2, and other arachidonate metabolites were less than 0.05%. These compounds were efficiently separated from 11-dehydro-thromboxane B2 by the immunoaffinity purification. This procedure also allowed the separation of 11-dehydro-thromboxane B2 from unidentified urinary and plasma substances which interfered with the radioimmunoassay. Validity of the results obtained by the radioimmunoassay was confirmed by GC/MS employing selected ion monitoring for quantification.

Enzyme immunoassay of thromboxane B2

An immunoassay for thromboxane B2 was developed in which the hapten molecule was labeled with beta-galactosidase. The immunoprecipitate formed after competition between enzyme-labeled and unlabeled thromboxane B2 was subjected to a fluorometric assay of beta-galactosidase. Thromboxane B2 was detectable in the range of 0.1-30 pmol. Both enzyme immunoassay and radioimmunoassay showed essentially the same cross-reactivities with other prostaglandins and their metabolites when the same antibody was used. Known amounts of thromboxane B2 were added to human plasma, and the sample was applied to an octadecyl silica column. The extract was analyzed by enzyme immunoassay to examine the correlation between the added (x) and measured (y) thromboxane B2 (y = 1.09x + 11.07 pmol/ml, r = 0.99). A satisfactory correlation was observed between radioimmunoassay (x) and enzyme immunoassay (y) (y = 0.92x + 4.64 pmol/ml, r = 0.96). The validity of enzyme immunoassay was also confirmed by gas chromatography-mass spectrometry of a dimethylisopropylsilyl ether derivative of thromboxane B2 methyl ester. The method was applicable to the assay of thromboxane B2 produced from endogenous precursor during thrombin-induced aggregation of human platelets.

TNFα-Dependent Induction of Cyclooxygenase-2 Mediated by NFκB and NF-IL6

MC3T3-E1 cell is an osteogenic line established from newborn mouse calvaria by Kodama and others.1 Our laboratory has demonstrated that the cells release prostaglandin (PG) E2 into the culture medium as an essentially sole arachionate metabolite, and the PGE2 production is stimulated markedly by the addition of EGF, TGFβ, epinephrine or various PGs.2–5 Several lines of evidence suggested that the increased PGE2 synthesis was attributed to the induction of cyclooxygenase enzyme by these compounds. Furthermore, we reported that the increased cyclooxygenase activity by PGs (PGF2α, PGE2, PGE1 and iloprost) was mostly attributed to the induction of cyclooxygenase-2 rather than cyclooxygenase-1.6 The quantification of c AMP and IP3 and the examination of the effects of various protein kinase inhibitors indicated that iloprost and PGE1 induced cyclooxygenase-2 through a pathway mediated by cAMP and A kinase. On the other hand, PGF2α induced cyclooxygenase-2 via PI turnover and protein kinase C. PGE2 was probably coupled with two or more pathways.

Enzyme immunoassay of 6-ketoprostaglandin F1α in a solid phase

Abstract A solid-phase enzyme immunoassay of 6-ketoprostaglandin F1α (a stable degradation product of prostaglandin I2) was developed in which the hapten molecule was labeled with β-galactosidase. The antiserum was bound to a polystyrene tube, and the enzyme-labeled and unlabeled 6-ketoprostaglandin F1/ga were allowed to react in a competitive manner with the immobilized antibody. The activity of the immunologically bound β-galactosidase was assayed by fluorometry, and correlated with the amount of unlabeled 6-ketoprostaglandin F1α According to a calibration curve 6-ketoprostaglandin F1α was detectable in a range of 30 fmol-10 pmol. When 6-ketoprostaglandin F1α was extracted from human serum by using an octadecylsilyl silica column (Sep-Pak C18) and the crude extract was subjected to the enzyme immunoassay, the content of 6-ketoprostagland F1α was 50–60 pmol / ml of human serum. An endogenous substance(s) which disturbed the immunoreaction and gave such an apparently high concentration of 6-ketoprostaglandin f1α was separated from the endogenous 6-ketoprostaglandin F1α by HPLC. With the purified 6-ketoprostaglandin F1α fraction there was a good correlation (r = 0.994) between enzyme immunoassay and radioimmunoassay. The validity of the enzyme immunoassay was confirmed by gas chromatography-selected ion monitoring.

Effects of aspirin DL-lysine on thrombin generation in unstable angina pectoris

To evaluate the effects of aspirin on thrombin generation in patients with unstable angina, plasma levels of thrombin-antithrombin III complex (TAT) as a new marker of thrombin generation and of 11-dehydro-thromboxane B2 (11-dehydro-TXB2) as an indicator of platelet activation were measured in 18 patients with unstable angina, including 8 patients with prolonged rest angina (> 15 minutes). Aspirin DL-lysine (900 mg) was administered intravenously to 9 of the 18 patients (aspirin group); the other 9 were not given aspirin during the first 24 hours of hospitalization (non-aspirin group). Clinical characteristics, angiographic features and medications other than aspirin were similar between the 2 groups. Levels of plasma TAT and 11-dehydro-TXB2 were significantly higher (p < 0.05) in patients with prolonged rest angina than in those without the condition (n = 10). In 5 patients with prolonged rest angina who received aspirin, plasma TAT levels (ng/ml) were significantly decreased (4.52 +/- 1.18 at baseline, 2.50 +/- 0.65 at 1 hour and 2.16 +/- 0.42 at 24 hours after aspirin administration, p < 0.01) with a significant decrease in plasma 11-dehydro-TXB2 levels. However, the reduction in TAT after aspirin administration was slight in patients without prolonged rest angina (n = 4). In contrast, levels of plasma TAT and 11-dehydro-TXB2 in the non-aspirin group remained unchanged during the study period. These results suggest that aspirin rapidly reduces thrombin generation through inhibition of platelet activity in patients with unstable angina with prolonged rest angina.

Microdetermination of 11-dehydrothromboxane B2 in human urine by gas chromatography selected-ion monitoring

Simple and effective purification for quantitation of trace amounts of 11-dehydrothromboxane B2 (11-dehydro-TXB2) in human urine by gas chromatography-selected-ion monitoring (GC-SIM) was achieved. The procedure is based on stepwise elution of the methyl ester derivative from a silica gel column with n-hexane-ethyl acetate (1:1) after washing the column with n-hexane-ethyl acetate (2:1). After the methyl ester has been converted into the corresponding dimethylisopropylsilyl ether, GC-SIM is carried out by monitoring the ion at m/z 539.32 for 11-dehydro-TXB2 and that at m/z 543.33 for its 18O2-labelled variant as an internal standard. The detection limit is 2 pg per injection with a signal-to-noise ratio of 5:1. The method was applied to the determination of 11-dehydro-TXB2 in human urine.

Enzyme immunoassay of human plasma 11-dehydrothromboxane B2

11-Dehydrothromboxane B2 (11-dhTXB2) is a proposed marker compound for thromboxane A2 formed in vivo. An enzyme immunoassay was established for determination of the plasma concentration of this compound. The assay was based on a horseradish peroxidase-linked immunoassay utilizing polyclonal anti-11-dhTXB2 antibody obtained from a rabbit, and enabled determination of 11-dhTXB2 in the range of 2 to 500 pg/tube with an IC50 of 36 pg. The cross-reactivities with TXB2, 2,3-dinor-TXB2 and other prostanoids were less than 0.05%. Validity of the enzyme immunoassay was confirmed by a radioimmunoassay utilizing a monoclonal antibody. The plasma 11-dhTXB2 was immunoaffinity-purified by one step using an immobilized monoclonal antibody. The mean plasma level of 11-dhTXB2 in six male volunteers was 4.0 +/- 0.3 pg/ml by this enzyme immunoassay.

Immunoaffinity Purification of Prostaglandin E2 and Leukotriene C4 Prior to Radioimmunoassay: Application to Human Synovial Fluid

When human synovial fluid as such was subjected to radioimmunoassays of prostaglandin E2 (PGE2) and leukotriene C4 (LTC4), there was no linear increase in PGE2 and LTC4 as the amount of synovial fluid was raised. For removal of substances thus disturbing the assay we developed a method of immunoaffinity purification of PGE2 and LTC4. A monoclonal antibody against PGE2 or LTC4 was coupled to BrCN-activated Sepharose 4B. When synovial fluid mixed with radiolabelled PGE2 or LTC4 was applied to the column of immobilized antibody, the ligand was adsorbed to the column and eluted with a mixture of methanol/water in a recovery of about 80%. The purified material showed a linearity between the amount of the sample and the value of radioimmunoassay. The one-step method was applied to synovial fluid from patients with rheumatoid arthritis, osteoarthritis and other joint diseases.

Novel derivatization and immunoextraction to improve microanalysis of 11-dehydrothromboxane B2 in human urine.

A new, highly selective procedure for the determination of 11-dehydrothromboxane B2 (11-dehydro-TXB2) in human urine is described. Following the addition of [19,19,20,20-2H4]11-dehydro-TXB2 as an internal standard, samples were extracted with an affinity column of anti-11-dehydro-TXB2. Conversion of the immunoextracted 11-dehydro-TXB2 into its 1-methyl ester-11-n-propylamide-9,12,15-tris-dimethylisopropylsilyl ether derivative was followed by gas chromatography-selected-ion monitoring. The mass spectrum of the 11-dehydro-TXB2 derivative was dominated by the base peak ion of [M-C3H7]+ at m/z 698, which accounted for more than 10% of the total ion current. A typical result showed that the immunoaffinity purification procedures provided an extremely clean alternative to more conventional methods of chromatographic fractionation, and that interfering substances from the urine matrix were almost entirely eliminated during the microanalysis.

[39] Enzyme immunoassay of PGF2α

Publisher Summary Enzyme immunoassay of prostaglandin (PG) F 2α is developed as a replacement of radioimmunoassay. In this method the antigen PGF 2α is labeled with an enzyme (β-galactosidase or alkaline phosphatase) rather than radioactivity, and the immune complex is measured by assaying enzyme activity. Because the application of this method to biological materials is now under investigation, this chapter describes the assay procedures with authentic PGF 2α . In assay procedures sequential saturation technique is used for competition among enzyme-labeled and free PGF 2α . Fluorescence intensity of the reaction product is determined against a freshly prepared standard of 0.25 or 0.50 M 4-methylumbelliferone using a Hitachi spectrofluorometer. The wavelengths used for excitation and emission are 360 and 450 nm, respectively. One unit of β-galactosidase is defined as the amount of enzyme that hydrolyzes 1μmol of 4-methylumbelliferyl-β-D-galactoside per minute under the standard assay conditions.